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Protocol for RNA extraction using Trizol reagent
Based on the user guide of Trizol reagent by Invitrogen.
Material
Contents and storage
| Contents | Cat. No. 15596026 (100 reactions) | Cat. No. 15596018 (200 reactions) | Storage |
|---|---|---|---|
| TRIzolTM Reagent | 100 mL | 200 mL | 15–30°C |
Required items which is not supplied.
| Item | Memo |
|---|---|
| Equipment | |
| Water bath or heat block at 55–60°C | |
| Reagents | |
| Isopropanol | |
| Ethanol, 75% | |
| RNase-free water of 0.5% SDS | |
| (Optional) RNase-free glycogen |
Input sample requirements
IMPORTANT!
Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.
| Sample type | Starting material per 1 mL of TRIzolTM Reagent |
|---|---|
| Tissues[1] | 50 ー 100 mg of tissue |
| Cells grown in monolayer | 1 × 10xx5 ー 1 × 10xx7 cells grown in monolayer in a 3.5–cm culture dish (10 cm2) |
| Cells grown in suspension | 5 ー 10 × 10xx6 cells from animal, plant, or yeasty origin or 1 × 10xx7 cells of bacterial origin |
| [1] Fresh tissues or tissues stored in RNAlaterTM Stabilization Solution (Cat. No. AM7020). | |
Procedural guidelines
- Perform all steps at room temperature (20–25°C) unless otherwise noted.
- Use cold TRIzolTM Reagent if the starting material contains high levels of RNase, such as spleen or pancreas samples.
- Use disposable, individually wrapped, sterile plastic ware and sterile, disposable RNase-free pipettes, pipette tips, and tubes.
- Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials.
- Always use proper microbiological aseptic techniques when working with RNA.
- Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.
Protocol
1 Lyse samples and separate phases
- Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.
Tissues:
- Add 1 mL of TRIzolTM Reagent per 50–100 mg of tissue to the sample and homogenize using a homogenizer.
Cell grown in monolayer:
- Remove growth media.
- Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.
- Pipet the lysate up and down several times to homogenize.
Cells grown in suspension:
- Pellet the cells by centrifugation and discard the supernatant.
- Add 0.75 mL of TRIzolTM Reagent per 0.25 mL of sample (5– 10 × 106 cells from animal, plant, or yeasty origin or 1 ×107 cells of bacterial origin) to the pellet.
Note: Do not wash cells before addition of TRIzolTM Reagent to avoid mRNA degradation.
- Pipet the lysate up and down several times to homogenize.
Note: The sample volume should not exceed 10% of the volume of TRIzolTM Reagent used for lysis.
STOPPING POINT Samples can be stored at 4°C overnight or at – 20°C for up to a year.
- (Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
- Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
- Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysis, then securely cap the tube.
- Incubate for 2–3 minutes.
- Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
- The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.
- Transfer the aqueous phase containing the RNA to a new tube. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
IMPORTANT! Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase.